In the United States, cardiovascular disease (CVD) accounts for approximately 1.4 million deaths annually, representing nearly 40% of all deaths. The projected cost of heart disease and stroke for 2007 is $431.8 billion. The discovery of new biomarkers will be critical for much-needed advances in screening, diagnosis, prognosis, prediction of therapeutic response and therapeutic monitoring for this devastating disease. The goal for Phase I of this Project is to optimize in human plasma a new proteomics technology, Differential Capture ProteomicsTM (DCP), which can identify the differences in protein composition between two biological samples. This technology also simultaneously creates affinity reagents for each of the identified difference proteins. A patent for DCP has been issued to Differential Proteomics Inc. The goal for a subsequent Phase II study is to apply DCP, newly optimized for human plasma, to the discovery of biomarkers of CVD using appropriate patient [plasma] samples to enable novel protein isolation and identification, novel affinity reagent creation, assay formulation, and clinical validation. Studies in our laboratory to optimize the DCP technology have successfully focused on a model system. However, the capabilities of DCP for CVD biomarker discovery in patient plasma samples have yet to be demonstrated with an optimization study. Such a study is proposed here, in which doped CVD biomarkers (representing a model CVD patient sample) will be tested for detectability at clinical sample level concentrations. The proposed study will use five specific CVD protein antigens, for which there are commercially available immunoassays. The success of this Phase I study will lead to a Phase II program for testing multiple CVD patient [plasma] samples for novel biomarker discovery, assay generation, and validation. Specific Aim 1: Confirm that three pooled Random Peptide Phage Libraries actually contain binding phage species against each of the five CVD antigens chosen. Specific Aim 2: Show how well Differential Capture Proteomics detects the differences between two human plasma samples, one of which is doped with the mixture of five CVD antigens. Specific Aim 3: Establish the specificity of the affinity capture reagents that are generated as part of the process. PUBLIC HEALTH RELEVANCE: Since Differential Capture Proteomics will be significantly more cost- and time-effective than current approaches, it potentially offers a breakthrough system for the integrated discovery, verification, and validation of biomarkers. The discovery of new biomarkers of cardiovascular disease will be critical for much-needed advances in screening, diagnosis, prognosis, prediction of therapeutic response and therapeutic monitoring for this devastating disease.